This invention relates to a process for measuring the degree of coloring of a color developing reagent to be reduced such as tetrazolium compounds in determination of the amount of hydrogen peroxide generated or peroxidase activity in clinical examinations.
Detection and determination of hydrogen peroxide (H.sub.2 O.sub.2) are very important not only in chemical experiments and industrial applications but also in widely used clinical examinations wherein determination of living body components is carried out by determining H.sub.2 O.sub.2 generated by enzymatic reactions. For example, the amounts of cholesterol, triglyceride, glucose, uric acid, phospholipids, bile acid, choline esterase, monoamine oxidase, guanase, etc., are measured by determining H.sub.2 O.sub.2 finally produced in individual detective (or determination) systems. Such methods are applied to diagnoses of diseases.
The most widely used method for determining H.sub.2 O.sub.2 is to use peroxidase and an oxidizable color reagent as a color developing component to develop a color, which is measured colorimetrically for indirectly determining the amount of desired components. Typical examples of the oxidizable color reagents used in this method are a combination of 4-aminoantipyrine and a phenolic compound or an N,N-disubstituted aniline compound; a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and an aniline compound; 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); triphenylmethane series leuco dyes; benzidine derivatives; diphenylamine derivatives; triarylimidazole derivatives; o-phenylenediamine; etc.
But when these oxidizable color reagents were used in determination of trace components in living body samples such as tisses, body fluid, etc., there took place negative errors in measured values due to an influence of a reduction reaction caused by a reductive substance such as ascorbic acid, bililubin, etc. present in a sample of living bodies. In order to remove such interfering substances, there have been proposed various methods, for example, the use of ascorbate oxidase, an iodate, copper ion-peroxidase-amino compound, etc. to remove ascorbic acid, and the use of potassium ferrocyanide, bilirubin oxidase, etc. to remove bilirubin. But such proposals had a merit and a demerit.
Further, the above-mentioned determination method of H.sub.2 O.sub.2 is often applied to measuring of individual lipids contained in lipoprotein fractions so as to stain individual lipids by color development by means of an enzymatic method using an oxidizable color reagent. For example, in the case of cholesterol, staining is conducted by using cholesterol oxidase to produce H.sub.2 O.sub.2, which is reacted with 4- aminoantipyrine and a phenolic compound in the presence of peroxidase (hereinafter referred to as "POD"). But such a method as using an oxidizable color reagent for staining of lipoprotein fractions has a serious problem in staining properties, since a part of produced dye is washed away in a washing step in aftertreatment.
On the other hand, peroxidase (POD) is used as an oxidizing catalyst in enzyme analysis methods using oxidases for producing hydrogen peroxide (H.sub.2 O.sub.2) in clinical examinations. Recently, POD has widely been used as a labeled enzyme in an immunoenzymatic staining method and enzyme immunoassay. Further, a method for specifically detecting trace amounts of proteins in a living body by using an enzyme (e.g. POD) labeled antibody and by combining electrophoresis with blotting operations has been noted as an epoch-making protein analysis method.
These immunological methods are excellent in specificity, so that application of these methods may be broadened more and more in the future.
The immunoenzymatic staining methods using POD include typically a peroxidase-antiperoxidase complex (PAP) method which is generally used in a histological immunoenzymatic staining method wherein an immune reaction is applied to tissue slices, an avidin-biotin-peroxidase labeling (ABC) method, etc. The final staining in these methods is based on a color reaction in a H.sub.2 O.sub.2 -oxidizable color reagent system applying POD activity.
The enzyme immunoassay using POD is a method comprising measuring POD activity of a POD labeled antibody bonded to a substance to be measured using a H.sub.2 O.sub.2 -oxidizable color reagent and obtaining the concentration of the substance to be measured from the activity value measured.
These methods mainly use oxidizable color reagents such as o-phenylenediamine, 3,3'-diaminobenzidine, 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole, etc. But these methods using the oxidizable color reagents sometimes cause negative errors in measured values by the influence of reducing substances such as ascorbic acid, bilirubin, etc. present in samples, when applied to determination of trace amounts of components present in living bodies. In order to remove these interfering substances, various proposals have been made; for example, ascorbic acid is removed by ascorbate oxidase and an iodate, and bilirubin is removed by potassium ferrocyanide and bilirubin oxidase. But such proposals had a merit and a demerit.
In various detecting and determining methods for various trace amounts of proteins by using electrophoresis and blotting operations in combination, a H.sub.2 O.sub.2 -oxidizable color reagent (such as 3,3'-diaminobenzidine, 4-chloro-1-naphthol, etc.) system is used for measuring activity of POD which is a labeled enzyme. But such methods are not satisfactory in sensitivity and color tone.
Further, many of these oxidizable color reagents are unstable and have a defect in that stained samples to be measured are faded during storage particularly in the histological immunoenzymatic staining method.
On the other hand, the use of a color producing reagent to be reduced is disclosed in European Patent Appln. Laid-Open No. 0100217 in the determination of super oxide anion. But such a method could not be applied to the determination of the amount of H.sub.2 O.sub.2 or peroxidase activity.